ThunderSTORM’s MFA option produces excellent results with recognising and separating overlapping EBCCD photon events, as shown in Fig \ref{fig_mfa}. For the USAF data containing an average of 150 photons/frame the photon count increases 2% with MFA enabled.

Centroiding was then applied to higher photon density fluorescence microscopy data of F-actin in a fixed BPAE cell labelled with Alexa-488 (Fig \ref{fig_cell}). The 6000-frame data set contains an average of 460 photons/frame. With 7-pixel PSF fitting radius and MFA enabled, the photon count increases 13.0% compared to using 2-pixel fitting radius, and 5.2% compared to 7-pixel fitting radius without MFA. Fitting of multiple PSFs is time-consuming and the processing time increases significantly with MFA enabled, as expected (see Table \ref{table:results}). However, for biological imaging the image acquisition time is a critical parameter, and the separation of overlapping events can potentially lead to significant reduction in image acquisition times.